Probing the Role of Rotational Dynamics in Cellular Transport

While it can generally be said that cellular function is critically dependent on the fidelity of cargo transport, processive transport is even more important in the axons and dendrites of neurons, where a cell must regulate populations of molecules on length scales that can range up to meters. Consequently, much effort has been made to investigate the translocation of cargoes in neurons and the properties of the motors responsible therein. Though biocompatible fluorophores have become increasingly powerful tools for study of motor-driven transport, they suffer from photobleaching and require bright illumination which can be toxic to live cells. Most conventional fluorescent approaches are further limited by the lack of oreintation information they provide. On the other hand, with the small diameter of neurites and the high levels of traffic they support through a crowded environment, orientation of the cargoes relative to the cytoskeletal tracks they are moving on can be vital. We present an experimental approach making use of dark field optical microscopy and gold nanorods as reporters of both lateral translocation as well as orientation of cargo in neurons. using relatively low illumination intensity, we can measure dynamics of single cargoes moving in the image plane and resolve changes in the azimuth and polar angles all at millisecond time scales. Furthermore, the gold nanorods can be specifically delivered to the cell body or axon terminal by culturing the neurons in microfluidic devices with separate chambers, enabling the investigator to resolve differences between retrograde and anterograde transport. The ability to track axonal transport with a high temporal and spatial dynamic range reveals several kinds of orientational changes of moving cargoes that correlate with transport dynamics, allowing more detailed inferences into changes in the activity of molecular motors.